ABSTRACT Recent efforts to identify and circumvent mechanisms of drug resistance point to the role of HER2 truncated isoforms, which lack the extracellular domain of the full-length protein targeted by trastuzumab. Understanding the biology of HER2 isoforms is crucial to developing new drugs personalized for individuals resistant to trastuzumab therapy. However, detection of these isoforms in paraffin-embedded tissue sections (FFPE) is impossible by conventional methods (Immunohistochemistry, IHC) due to the lack of isoform-specific antibodies and the loss of epitope integrity during tissue processing. To overcome these barriers, the goal of this project is to develop a robust tool that will both allow retrospective studies on HER2 isoforms and enable isoform screening on a patient-to-patient basis to tailor breast cancer treatment. Our efforts fill a major gap in cancer research: the limited ability to scrutinize tissue sections for protein targets that either lack highly specific antibody probes (analytical) or lose epitope integrity during tissue processing (pre- analytical). We introduce a whole-tissue western blot (wtWestern) that creates a novel 3D protein ?imprint? of the tissue section for stable storage and downstream antibody probing. The unique dual-functionality wtWestern electrophoretically resolves proteins (extracted from a 2D tissue section) along the depth (z-axis) of a supporting photoactive hydrogel layer. Exposure to UV light covalently links protein to the hydrogel. We benchmark the fixative-free imprint for biobanking suitability, apply the wtWestern to measurement of proteins (including isoforms) in archived frozen or FFPE tissue sections, and maximize tissue utility by enabling detection of 20+ proteins for cancer sub-type classification. This innovative wtWestern is expected to maximize the quality and utility of biospecimens for downstream analyses ? critically important to retrospective studies that form the basis for both cancer diagnostics/prognostics and personalized medicine.